TcI Isolates of Trypanosoma cruzi Exploit the Antioxidant Network for Enhanced Intracellular Survival in Macrophages and Virulence in Mice.

Trypanosoma cruzi species is categorized into six discrete typing units (TcI to TcVI) of which TcI is most abundantly noted in the sylvatic transmission cycle and considered the major cause of human disease. In our study, the TcI strains Colombiana (COL), SylvioX10/4 (SYL), and a cultured clone (TCC) exhibited different biological behavior in a murine model, ranging from high parasitemia and symptomatic cardiomyopathy (SYL), mild parasitemia and high tissue tropism (COL), to no pathogenicity (TCC). Proteomic profiling of the insect (epimastigote) and infective (trypomastigote) forms by two-dimensional gel electrophoresis/matrix-assisted laser desorption ionization-time of flight mass spectrometry, followed by functional annotation of the differential proteome data sets (≥2-fold change, P < 0.05), showed that several proteins involved in (i) cytoskeletal assembly and remodeling, essential for flagellar wave frequency and amplitude and forward motility of the parasite, and (ii) the parasite-specific antioxidant network were enhanced in COL and SYL (versus TCC) trypomastigotes. Western blotting confirmed the enhanced protein levels of cytosolic and mitochondrial tryparedoxin peroxidases and their substrate (tryparedoxin) and iron superoxide dismutase in COL and SYL (versus TCC) trypomastigotes. Further, COL and SYL (but not TCC) were resistant to exogenous treatment with stable oxidants (H2O2 and peroxynitrite [ONOO(-)]) and dampened the intracellular superoxide and nitric oxide response in macrophages, and thus these isolates escaped from macrophages. Our findings suggest that protein expression conducive to increase in motility and control of macrophage-derived free radicals provides survival and persistence benefits to TcI isolates of T. cruzi.

American tegumentary leishmaniasis: T-cell differentiation profile of cutaneous and mucosal forms-co-infection with Trypanosoma cruzi.

American tegumentary leishmaniasis displays two main clinical forms: cutaneous (CL) and mucosal (ML). ML is more resistant to treatment and displays a more severe and longer evolution. Since both forms are caused by the same Leishmania species, the immunological response of the host may be an important factor determining the evolution of the disease. Herein, we analyzed the differentiation and memory profile of peripheral CD4(+) and CD8(+) T lymphocytes of patients with CL and ML and their Leishmania-T. cruzi co-infected counterparts. We measured the expression of CD27, CD28, CD45RO, CD127, PD-1 and CD57, together with interferon-γ and perforin. A highly differentiated phenotype was reflected on both T subsets in ML and preferentially on CD8(+) T cells in CL. A positive trend toward a higher T differentiation profile was found in T. cruzi-infected CL and ML patients as compared with Leishmania single infections. Association between CD8(+) T-cell differentiation and illness duration was found within the first year of infection, with progressive increase of highly differentiated markers over time. Follow-up of patients with good response to therapy showed predominance of early differentiated CD8(+) T cells and decrease of highly differentiated cells, while patients with frequent relapses presented the opposite pattern. CD8(+) T cells showed the most striking changes in their phenotype during leishmaniasis. Patients with long-term infections showed the highest differentiated degree implying a relation between T differentiation and parasite persistence. Distinct patterns of CD8(+) T differentiation during follow-up of different clinical outcomes suggest the usefulness of this analysis in the characterization of Leishmania-infected patients.

[Miltefosine versus meglumine antimoniate in the treatment of mucosal leishmaniasis]. / Miltefosina versus antimoniato de meglumina en el tratamiento de la leishmaniasis mucosa.

The conventional treatment for tegumentary leishmaniasis is meglumine antimoniate, which needs parenteral administration, has increased therapeutic failure, and produces serious adverse effects, justifying the search for therapeutic alternatives. We report here the preliminary results of a phase II clinical trial in patients with mucosal leishmaniasis, in which the efficacy of oral miltefosine versus the antimonial compound was assessed. The evaluation of response to the treatment was performed by monitoring with nasopharyngeal video-fibroscopy, using a score of mucosal injury severity for patients at each follow-up point. We found no significant differences so far between the number of patients cured with miltefosine or conventional chemotherapy. The favorable results of this study suggest that miltefosine could be an effective and safe oral therapeutic alternative in the region.

Polymorphism-specific PCR enhances the diagnostic performance of American tegumentary leishmaniasis and allows the rapid identification of Leishmania species from Argentina.

BACKGROUND: The diagnosis of the leishmaniases poses enormous challenges in Argentina. The Polymorphism-Specific PCR (PS-PCR) designed and validated in our laboratories has been proven effective for typifying the Leishmania genus from cultured material. Here we evaluated the performance of this method in the diagnosis of American tegumentary leishmaniasis (ATL) and the rapid identification of Leishmania spp. directly from clinical specimens. METHODS: A total of 63 patients from northwestern Argentina, with cutaneous or mucocutaneous lesions, underwent an ATL diagnosis protocol which included clinical examination, Leishmanin skin test, and microscopic examination of dermal smears. In addition, we performed PS-PCR on DNA directly extracted from the specimens scraped from the lesions. RESULTS: Out of the 63 patients, 44 were classified as ATL cases and 19 as non-ATL cases. The diagnostic sensitivity of the microscopic analysis of dermal smears and PS-PCR individually were 70.5% and 81%, respectively. When performing both tests in parallel, this parameter increased significantly to 97.6% (p = 0.0018). The specificities, on the other hand, were 100%, 84.2%, and 83.3% for the combination, respectively (p > 0.05). Using the PS-PCR analysis we successfully identified the Leishmania spp. in 31 out of the 44 ATL cases. Twenty-eight (90.3%) cases were caused by L. (V.) braziliensis, two (6.5%) by L. (V.) guyanensis, and one (3.2%) by L. (V.) panamensis. CONCLUSIONS: The efficacy of the ATL diagnosis was significantly improved by combining the dermal smear examination with a PS-PCR analysis. Our strategy allowed us to reach the diagnosis of ATL with high accuracy regarding the species of the etiological agent in 70.5% of the cases. Moreover, we diagnosed two cases of the disseminated cutaneous form caused by L. (V.) braziliensis and a cutaneous case due to L. (V.) panamensis infection, both findings reported for the first time in Argentina.

[Comparison of nucleic acid extraction efficiency using different commercial kits and qPCR. Effect of inhibitors]. / Comparación de la eficiencia de extracción de ácidos nucleicos utilizando distintos kits comerciales y empleando la qPCR. Efecto de las sustancias inhibidoras.

The detection of specific nucleic acid (NA) sequences by PCR has revolutionized the biological and medical sciences. Real-time PCR (qPCR) opened up the possibility of obtaining quantitative results. NA extraction is a decisive step prior to qPCR since it may produce either the removal or co-extraction of inhibitory substances of the enzymatic reaction, which in turn affects the amplification efficiency. In the present work we compared the commercial NA extraction kits from Qiagen, Invitrogen and Macherey-Nagel, which were used to extract DNA from mice blood artificially infected with Trypanosoma cruzi and PP7 RNA, Pseudomonas aeruginosa bacteriophage, in spiked aqueous matrices. NA recovery efficiency in samples without inhibitors was similar for the three extraction kits. However, the Invitrogen kit was the only one that remained unaffected in the presence of inhibitors in the samples.

Histologic and histomorphometric study of bone repair under acute Trypanosoma cruzi infection in rats.

Trypanosoma cruzi (T cruzi) is an intracellular protozoan pathogen that causes American trypanosomiasis (Chagas disease). The aim of this study was to evaluate the histopathological effects of acute infection by T. cruzi on bone repair, Wistar rats were used throughout. The animals were assigned to two groups: Control Group (CG n =20) and Experimental Group (EG n = 20). All the animals were anesthetized, at to the first lower right molar was extracted. The EG animals were inoculated subcutaneously at to with 0.1 mL of 10 trypomastigotes of the virulent strain Tulahuen of T. cruzi. The CG animals were administered an equivalent volume ofsaline solution subcutaneously. The animals in both groups were euthanized at 15 days post-infection and tooth extraction. The mandibles were resected, fixed informalin solution, radiographed, decalcified and embedded in paraffin. Bucco-lingually oriented sections were obtained at the level of the mesial tooth socket of the first lower molar and stained with hematoxylin-eosin. Total alveolar volume (TV) and bone volume (TBV/TV) in the apical third of the tooth socket were evaluated histomorphometrically. The histological analysis revealed an alteration in post-extraction bone tissue repair in animals infected by T. cruzi. A reduction in osteogenic activity was observed concomitant with a rise in quiescent and eroded bone surfaces. Histomorphometric evaluation revealed a significant reduction (19%) in total alveolar volume (TV) and bone volume (TBV/TV) (24%) in the apical third of the tooth socket in animals infected with T. cruzi in comparison to non-infected animals (p<0.05). The results obtained using this experimental model showed decreased osteogenesis in bone tissue repair under acute Trypanosoma cruzi infection in rats.

[Alterations of immunological parameters in an autochthonous case of visceral leishmaniasis from the Northwest of Argentina]. / Afectación de parámetros inmunológicos en un caso de leishmaniasis visceral autóctono del Noroeste Argentino.

We report the alterations of immunological parameters of a patient with visceral leishmaniasis caused by Leishmania (Leishmania) infantum from the Northwest of Argentina during active disease and after achieving clinical recovery. We first demonstrated elevated amounts of IFN-y, IL-10, B-cell activating factor (BAFF) and IgG in plasma during active disease, which returned to control values after recovery. In relation to T cell profile, we measured CD27, CD28, CD45RO, CD57 and perforin. We found a highly differentiated phenotype, preferentially in active disease and among CD8+ T cells, consisting in increased numbers of late differentiated and terminal effector cells. Although this highly differentiated CD8+ T cell phenotype persisted after recovery, a clear increase of central memory cells was recorded for both T subsets at that point, suggesting signs of reversion toward a less differentiated profile. The composition of the B cell compartment was slightly modified during active disease. Herein we document the global impact of severe visceral leishmaniasis on immunological parameters, which tend to revert upon clinical recovery, suggesting signs of immune restoration accompanying clinical improvement. The evaluated parameters could eventually be used as biomarkers of clinical evolution of visceral leishmaniasis.

En el presente trabajo informamos la afectación de parámetros inmunológicos durante la etapa grave de la infección y luego de alcanzar la recuperación clínica en un paciente autóctono del Noroeste argentino con leishmaniasis visceral causada por Leishmania (Leishmania) infantum. Detectamos concentraciones plasmáticas elevadas de interferón-y, interleuquina 10, IgG y BAFF (B-cell activating factor) durante la enfermedad activa, que se normalizaron luego de la recuperación clínica. En relación al perfil de diferenciación y memoria de las células T, clasificamos las células según la expresión de CD27, CD28, CD45RO, CD57 y perforina. Encontramos un fenotipo altamente diferenciado analizando la población de linfocitos T CD8+, con porcentajes aumentados de células T de diferenciación tardía y efectoras terminales. Si bien el fenotipo T CD8+ persistió luego de la recuperación clínica, pudimos observar un claro aumento de células T de memoria central en ese punto de estudio, sugiriendo signos de una posible reversión hacia un perfil T menos avanzado. El compartimiento de células B CD19+ mostró cambios más leves en la composición de las subpoblaciones de memoria. Documentamos el compromiso global de parámetros inmunológicos en la etapa grave de la leishmaniasis visceral que tienden a revertir luego de la recuperación, sugiriendo posibles signos de reconstitución inmune acompañando a la mejoría clínica. Los parámetros evaluados podrían ser útiles como biomarcadores de la evolución clínica de la enfermedad.

High performance of an enzyme linked immunosorbent assay for American tegumentary leishmaniasis diagnosis with Leishmania (Viannia) braziliensis amastigotes membrane crude antigens.

The diagnosis of American tegumentary leishmaniasis (ATL) still requires the design of more effective tools. Leishmania (Viannia) braziliensis is the causal agent of the 90% of Argentinean ATL cases. Considering the current knowledge, an ELISA based crude antigen (CA) for the diagnosis was designed. Ninety-nine subjects diagnosed as ATL, 27 as no-ATL, and 84 donors from non-ATL-endemic areas were included in this study. The current ATL diagnosis was based four techniques, dermal smear microscopic examination (parasitological test), PCR, Leishmanin skin test, and clinical records. We obtained CA extracts from promastigotes and amastigotes from macrophage cultures of different zymodemes of endemic Leishmania species circulating in the study area. Crude antigens from the 'local' main zymodeme of L. (V.) braziliensis showed the highest reactivity against anti-Leishmania antibodies compared to the other included species. The CA of amastigotes of this zymodeme was 3.4 fold more reactive than promastigotes one. Moreover, amastigote-membrane CA (MCA) were 3.6 fold more reactive than the soluble antigens. The MCA-ELISA reached a sensitivity and specificity of 98% (CI = 94.7%-100%) and 63.6% (53.9-73.1), respectively. When anti-Trypanosoma cruzi reactive sera were excluded, the specificity reached 98.4% (94.4-100), while the sensitivity was similar, with a positive predictive value (PV) of 98.6% (94.6-100) and negative PV of 96.3% (91.6-100). The performance of the MCA-ELISA results strongly contribute to the final diagnostic decision, since a non-reactive serological result almost discards the suspected ATL, because of its high negative PV. The developed MCA-ELISA showed a high diagnostic performance, which makes it a good candidate for ATL diagnosis, for seroprevalence studies, or for monitoring treatments efficacy.

Impairment of infectivity and immunoprotective effect of a LYT1 null mutant of Trypanosoma cruzi.

Trypanosoma cruzi infection of host cells is a complex process in which many proteins participate but only a few of these proteins have been identified experimentally. One parasite factor likely to be involved is the protein product of LYT1, a single-copy gene cloned, sequenced, and characterized by Manning-Cela et al. (Infect. Immun. 69:3916-3923, 2001). This gene was potentially associated with infectivity, since the deletion of both LYT1 alleles in the CL Brenner strain (the wild type [WT]) resulted in a null mutant T. cruzi clone (L16) that shows an attenuated phenotype in cell culture models. The aim of this work was to characterize the infective behavior of L16 in the insect vector and murine models. The infection of adult Swiss mice with 10(3) trypomastigotes of both clones revealed a significant reduction in infective behavior of L16, as shown by direct parasitemia, spleen index, and quantitation of tissue parasite burden, suggesting the loss of virulence in the null mutant clone. Although L16 blood counts were almost undetectable, blood-based PCRs indicated the presence of latent and persistent infection during all of the study period and epimastigotes were reisolated from hemocultures until 12 months postinfection. Nevertheless, virulence was not restored in L16 by serial passages in mice, and reisolated parasites lacking the LYT1 gene and bearing the antibiotic resistance genes revealed the stability of the genetic manipulation. Histopathological studies showed a strong diminution in the muscle inflammatory response triggered by L16 compared to that triggered by the WT group, consistent with a lower tissue parasite load. A strong protection against a virulent challenge in both L16- and WT-infected mice was observed; however, the immunizing infection by the genetically modified parasite was highly attenuated.

Use of kDNA-based polymerase chain reaction as a sensitive and differentially diagnostic method of American Tegumentary Leishmaniasis in disease-endemic areas of northern Argentina.

We evaluated the polymerase chain reaction (PCR) for the diagnosis of endemic American Tegumentary Leishmaniasis in Salta, Argentina. Diverse Leishmania species, coexistence of mycotic and varicose ulcers, and high endemicity T. cruzi, represent diagnostic challenges in the region. We performed a simplified PCR using sensitive, generic primers on samples obtained by a non-invasive method. We tested different culture types and clinical specimens with other microorganisms that induce leishmaniasis-like lesions. The PCR had a sensitivity and specificity of 100%. Forty-five patients with presumptive leishmaniasis were compared to the PCR, smears, and the Montenegro skin test (MST). In the same population, the PCR had an increased sensitivity, detecting 25 of 45 cases compared with 16 of 45 for smears and had a higher sensitivity in detecting mucocutaneous lesions. Diagnosis by PCR was supported by clinical presentation, positive MST results, compatible epidemiology, and in some cases histopathologic results or isolation of parasites by culture. These findings indicate the convenience of incorporating this PCR into diagnostic strategies for detecting leishmaniasis in northern Argentina.

Are cytochrome B gene sequencing and polymorphism-specific polymerase chain reaction as reliable as multilocus enzyme electrophoresis for identifying Leishmania spp. from Argentina?

Recently, two techniques, polymerase chain reaction (PCR) amplification and sequencing of cytochrome b gene (cyt b gene sequencing) and polymorphism-specific PCR (PS-PCR) were recommended for Leishmania species identification. Before this study, however, the accuracy of these methods had not been tested against the multilocus enzyme electrophoresis, the current gold standard technique on this task. Therefore, a trial was done for the first time to compare the results obtained by these techniques, using 17 Argentinean Leishmania stocks in independent assays. For all the stocks examined, the same results at species level were obtained by the three techniques. Among them, 14 were assigned to L. (Viannia) braziliensis, and three to L. (V.) guyanensis. The two techniques, cyt b gene sequencing and PS-PCR, were able to distinguish between all the proven species responsible for leishmaniases in Argentina. Thus, both techniques were validated and could be used independently for the species designation of Leishmania parasites in the country.

Clinical and epidemiological features of leishmaniasis in northwestern-Argentina through a retrospective analysis of recent cases.

Leishmaniasis is a parasitic disease caused by hemoflagellates of the genus Leishmania and is transmitted to humans by the bite of infected phlebotomine sandflies. Depending on the Leishmania species, the disease has different clinical forms including cutaneous, mucocutaneous, and visceral manifestations. Previous studies performed in endemic zones of northwestern-Argentina, during epidemic outbreaks, have been important for detecting patients suffering from the acute phase of the disease, but have not given a complete representation of the clinical and epidemiological features in the region. Furthermore, due to the resurgence of leishmaniasis worldwide and in particular the large increase of international tourism to the region, it seems pertinent to update the current epidemiological and clinical profile of leishmaniasis in northwestern-Argentina. Here we present a retrospective analysis of 95 Leishmania positive cases, presenting between 2000 and 2014. Patients were derived from hospitals and diagnosed in our lab at the University of Salta, located in a non-endemic area in Salta, Argentina. We detected numerous extensive mucocutaneous cases (34/95, 35.8%) distinct from mucosal affected patients, some instances originating in locations with no previously reported human cases. Additionally patients suffering from concomitant diseases, besides leishmaniasis, were assessed. These included Chagas disease, syphilis, deep mycoses, tuberculosis, toxoplasmosis and intestinal parasitosis. This study updates the clinical and epidemiological features of leishmaniasis in northwestern-Argentina, and discusses the implications and management strategy for patients who acquire the disease in this region.

Species assignation of Leishmania from human and canine American tegumentary leishmaniasis cases by multilocus enzyme electrophoresis in North Argentina.

Sixteen Leishmania stocks, 15 isolated from patients with cutaneous (CL), mucocutaneous (MCL), or recurrent cutaneous leishmaniasis, plus one from a dog with CL in Salta and Corrientes Provinces, Argentina, were studied by multilocus enzyme electrophoresis. Thirteen of the stocks from humans were grouped in two zymodemes; nine termed as KMS 1, four as KMS 2, and assigned to Leishmania (Viannia) braziliensis. Two additional stocks from CL cases expressed a KMS 4 enzyme profile, corresponding to L. (V.) guyanensis. Although the parasites from the dog were also assigned to L. (V.) braziliensis, its zymodeme, KMS 3, was not expressed in any of the current human isolates. The characterization of Leishmania from a dog was done for the first time in Argentina. The importance of the intraspecific polymorphism in the induction of clinical forms and in the host-reservoir concept is briefly discussed, based on the zymodeme data of isolates from humans and dogs. The presence of L. (V.) guyanensis was confirmed in the country.

Early diagnosis of congenital Trypanosoma cruzi infection using PCR, hemoculture, and capillary concentration, as compared with delayed serology.

Congenital Trypanosoma cruzi infection is a highly pathogenic and underreported condition. Early recognition is essential for effective treatment. Umbilical chord blood from newborns (n = 302) to infected mothers was analyzed with microhematocrit, hemoculture, and PCR methods. Each subject was then followed serologically. In calibrated suspensions of T. cruzi in blood, the sensitivity of PCR was 27-fold higher than hemoculture. However, this advantage was not reflected during routine testing of samples from maternities, partly because of the uneven distribution of few parasites in small samples. Levels of detection of congenital infection were 2.9% (8/272) for microhematocrit, 6.3% (18/287) for hemoculture, 6.4% (15/235) for PCR, and 8.9% (27/302) for cumulated results. Evaluation against the standard of delayed serology indicates that the regular application of PCR, hemoculture, and microhematocrit to blood samples allows the rapid detection of about 90% of the congenitally infected newborns, in samples that can be obtained before the mother and child leave the maternity ward.

Afectación de parámetros inmunológicos en un caso de leishmaniasis visceral autóctono del Noroeste Argentino / Alterations of immunological parameters in an autochthonous case of visceral leishmaniasis from the Northwest of Argentina

Resumen En el presente trabajo informamos la afectación de parámetros inmunológicos durante la etapa grave de la infección y luego de alcanzar la recuperación clínica en un paciente autóctono del Noroeste argentino con leishmaniasis visceral causada por Leishmania (Leishmania) infantum. Detectamos concentraciones plasmáticas elevadas de interferón-γ, interleuquina 10, IgG y BAFF (B-cell activating factor) durante la enfermedad activa, que se normalizaron luego de la recuperación clínica. En relación al perfil de diferenciación y memoria de las células T, clasificamos las células según la expresión de CD27, CD28, CD45RO, CD57 y perforina. Encontramos un fenotipo altamente diferenciado analizando la población de linfocitos T CD8+, con porcentajes aumentados de células T de diferenciación tardía y efectoras terminales. Si bien el fenotipo T CD8+ persistió luego de la recuperación clínica, pudimos observar un claro aumento de células T de memoria central en ese punto de estudio, sugiriendo signos de una posible reversión hacia un perfil T menos avanzado. El compartimiento de células B CD19+ mostró cambios más leves en la composición de las subpoblaciones de memoria. Documentamos el compromiso global de parámetros inmunológicos en la etapa grave de la leishmaniasis visceral que tienden a revertir luego de la recuperación, sugiriendo posibles signos de reconstitución inmune acompañando a la mejoría clínica. Los parámetros evaluados podrían ser útiles como biomarcadores de la evolución clínica de la enfermedad.

Abstract We report the alterations of immunological parameters of a patient with visceral leishmaniasis caused by Leishmania (Leishmania) infantum from the Northwest of Argentina during active disease and after achieving clinical recovery. We first demonstrated elevated amounts of IFN-γ, IL-10, B-cell activating factor (BAFF) and IgG in plasma during active disease, which returned to control values after recovery. In relation to T cell profile, we measured CD27, CD28, CD45RO, CD57 and perforin. We found a highly differentiated phenotype, preferentially in active disease and among CD8+ T cells, consisting in increased numbers of late differentiated and terminal effector cells. Although this highly differentiated CD8+ T cell phenotype persisted after recovery, a clear increase of central memory cells was recorded for both T subsets at that point, suggesting signs of reversion toward a less differentiated profile. The composition of the B cell compartment was slightly modified during active disease. Herein wedocument the global impact of severe visceral leishmaniasis on immunological parameters, which tend to revert upon clinical recovery, suggesting signs of immune restoration accompanying clinical improvement. The evaluated parameters could eventually be used as biomarkers of clinical evolution of visceral leishmaniasis.

Trypanosoma cruzi diversity in the Gran Chaco: mixed infections and differential host distribution of TcV and TcVI.

The transmission cycles of Trypanosoma cruzi in the Gran Chaco are complex networks involving domestic and wild components, whose interrelationships are not well understood. Knowing the circuit of transmission of the different Discrete Typing Units (DTUs) of T. cruzi in the complex environment of the Chaco region is relevant to understanding how the different components (reservoirs, vectors, ecotopes) interact. In the present study we identified the DTUs infecting humans and dogs in two rural areas of the Gran Chaco in Argentina, using molecular methods which avoid parasite culture. Blood samples of humans and dogs were typified by PCR-DNA blotting and hybridization assays with five specific DNA probes (TcI, TcII, TcIII, TcV and TcVI). PCR analyses were performed on seropositive human and dog samples and showed the presence of T. cruzi DNA in 41.7% (98/235) and 53% (35/66) samples, respectively. The identification of infective DTUs was determined in 83.6% (82/98) and 91.4% (32/35) in human and dog samples, respectively. Single infections (36.7% - 36/98) and a previously not detected high proportion of mixed infections (47.9% - 47/98) were found. In a 15.3% (15/98) of samples the infecting DTU was not identified. Among the single infections TcV was the most prevalent DTU (30.6% - 30/98) in human samples; while TcVI (42.8% - 15/35) showed the highest prevalence in dog samples. TcV/TcVI was the most prevalent mixed infection in humans (32.6% - 32/98); and TcI/TcVI (14.3% - 5/35) in dogs. Significant associations between TcV with humans and TcVI with dogs were detected. For the first time, the presence of TcIII was detected in humans from this region. The occurrence of one human infected whit TcIII (a principally wild DTU) could be suggested the emergence of this, in domestic cycles in the Gran Chaco.

Experimental evidence of biological interactions among different isolates of Trypanosoma cruzi from the Chaco Region.

Many infectious diseases arise from co-infections or re-infections with more than one genotype of the same pathogen. These mixed infections could alter host fitness, the severity of symptoms, success in pathogen transmission and the epidemiology of the disease. Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits a high biological variability often correlated with its genetic diversity. Here, we developed an experimental approach in order to evaluate biological interaction between three T. cruzi isolates belonging to different Discrete Typing Units (DTUs TcIII, TcV and TcVI). These isolates were obtained from a restricted geographical area in the Chaco Region. Different mixed infections involving combinations of two isolates (TcIII + TcV, TcIII + TcVI and TcV + TcVI) were studied in a mouse model. The parameters evaluated were number of parasites circulating in peripheral blood, histopathology and genetic characterization of each DTU in different tissues by DNA hybridization probes. We found a predominance of TcVI isolate in blood and tissues respect to TcIII and TcV; and a decrease of the inflammatory response in heart when the damage of mice infected with TcVI and TcIII + TcVI mixture were compared. In addition, simultaneous presence of two isolates in the same tissue was not detected. Our results show that biological interactions between isolates with different biological behaviors lead to changes in their biological properties. The occurrence of interactions among different genotypes of T. cruzi observed in our mouse model suggests that these phenomena could also occur in natural cycles in the Chaco Region.

Genetic and clinical characterization of canine leishmaniasis caused by Leishmania (Leishmania) infantum in northeastern Argentina.

Leishmaniases comprise zoonotic diseases caused by protozoan flagellates of the Leishmania genus. They are endemic to South America, and the visceral form has been recently reported in Argentina. Dogs can play different roles in the Leishmania transmission cycles, depending mainly on the species of parasite involved. Here we focused on the clinical characterization of canine leishmaniasis (CanL) in Northeast Argentina and on the molecular typing of its etiological agent. The nested polymerase chain reaction and sequence analysis of the Leishmania cytochrome b (cyt b) gene was performed on DNA templates purified from lymph nodes, bone marrow or spleen aspirates obtained from 48 dogs previously diagnosed by the observation of Leishmania amastigotes on smears from these aspirates. Their clinical and epidemiological data were also recorded. Systemic abnormalities were observed in 46 subjects (95.8%), most frequently lymphadenopathy, and emaciation (89.6 and 75%). Furthermore, 87% also presented tegumentary abnormalities, such as alopecia (54.2%) or secondary skin lesions (47.9%), among others. Twenty three dogs were positive for cyt b amplification. The sequence analysis showed the presence of two genotypes, LiA1 and LiA2, assigned to Leishmania (Leishmania) infantum, with 99.9 and 100% homology with the reference strain MHOM/TN/80/IPT1 respectively. LiA1 was identified in 18 cases (78.3%) and LiA2 in five (21.7%). Two cyt b variants of L. (L.) infantum were incriminated as the causative agents of CanL cases from three cities: Posadas, Garupá, and Ituzaingó. All three cities are located in the northeastern area of the country, where these parasites seem to be spreading in urban areas.